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[81] Intracellular distribution, purification, and properties of dihydrofolate synthetase from pea seedlings
[81]
DIHYDROFOLATE
S Y N T H E T A S E F R O M PEA S E E D L I N G S
581
casei and S. f a e c i u m and suggests that its site of activity in the latter
organism is dihydrofolate synthetase. The eight compounds in the table and the substrate itself were all tested for inhibitory activity in their oxidized states. In no instance was inhibition observed. It is clearly mandatory that the 7- and 8-positions of the pteridine moiety be reduced to achieve binding of substrate and inhibitor to the enzyme. To confirm the kinetic behavior of the enzyme toward these newly synthesized pteroate analogs, independent experiments were carried out with a constant concentration of each drug and at least five levels of substrate concentration (i.e., dihydropteroic acid). From such velocity data, LineweaverBurk kinetic plots were obtained for each of the active compounds. The inhibitions exerted by compounds 1-4 of the table are found to be competitive in nature. The apparent K~ value for each compound is the result of at least five experiments, and it was observed experimentally that the kinetic data were reproducible to within 1%.
[81] I n t r a c e l l u l a r D i s t r i b u t i o n , P u r i f i c a t i o n , a n d Properties of Dihydrofolate Synthetase from Pea Seedlings By KAZUO IWAI, MASAMICHI IKEDA, and MASAHIRO KOBASHI
7,8-Dihydropteroate + L-glutamate+ATP Mn'+K+ (orM,'n )7,8-dihydrofolate+ADP +Pl
Assay Method Principle. The assay for dihydrofolate synthetase activity is based on the microbiological assay with Lactobacillus casei ATCC 7469 which is described by Iwai et al. 1 7,8-Dihydropteroic acid used as substrate is prepared from pteroic acid by reduction with sodium dithionite as described by Futterman ~ for reduction of folic acid to 7,8-dihydrofolic acid. Reagents
Tris-HCl buffer, 0.1 M, pH 8.8 MgSO4, 5 mM K2804, 50 mM L-Glutamic acid, 5 mM i K. lwai, O. Okinaka, and H. Yokomizo,Vitamins 35, 387 (1967). 2 S. Futterman,J. Biol. Chem. 228, 1031 (1957). METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
582
PTERIDINES, ANALOGS, AND PTER1N COENZYMES
[81]
ATP, 5 mM Dihydropteroic acid, 50 p~M 2-Mercaptoethanol, 50 mM Procedure. 3 The above reagents are added to a tube in a total volume of 1.0 ml. The reaction is started by the addition of the enzyme. After the incubation at 37° for 30 min, the reaction is stopped by heating the whole in a boiling water bath for 1 min. An aliquot of the reaction mixture is added to the single basal medium for folic acid in a final volume of 10 ml. Definition o f a Unit. One unit of enzyme is that amount required for the formation of 0.1 nmol of folate equivalent under the above conditions. Protein is determined by the method of Lowry et al.4 with bovine serum albumin as the standard. Isolation Procedure for Cell Particles 3 Fifty grams of 6-day-old pea seedlings (Pisum sativum L. var. Alaska) are homogenized in a mortar with 100 ml of isolation medium containing 0.5 M sucrose, 50 mM Tris-HCl buffer, pH 7.5, and 50 mM 2-mercaptoethanol. The homogenate is squeezed through four layers of gauze and then centrifuged by the modified method of Fujiwara and Iida 5 successively at 100 g for 5 min (debris and nuclei), at 1000 g for 12 rain (chloroplasts), at 20,000 g for 30 rain (broken chloroplasts and mitochondria), and at 105,000 g for 90 min (microsomes). The final supernatant is used as the soluble fraction. Each precipitated fraction is washed with the isolation medium and suspended in 10 mM Tris-HC1 buffer, pH 7.5, containing 50 mM 2-mercaptoethanol (the suspending buffer). The subcellular localization of the enzyme is given in Table I. Purification Procedure Principle. The enzyme can be partially stabilized in the presence of 0.8-2 M ammonium sulfate and 50 mM 2-mercaptoethanol, although the enzyme in cell-free extracts from whole pea seedling is quite unstable, e In contrast, the enzyme in the mitochondrial fraction is fairly stable and easily solubilized by more than 95% only when in the suspending buffer. 3 M. l k e d a and K. Iwai, J. Nutr. Sci. Vitaminol. 21, 1 (1975). 4 0 . H. L o w r y , N. J. R o s e b r o u g h , A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A. Fujiwara and S. Iida, J. Sci. Soil Manure, Jpn. 31, 467 (1960). e M. Ikeda and K. Iwai, Plant Cell Physiol. 11,639 (1970).
[81]
DIHYDROFOLATE SYNTHETASE FROM PEA SEEDLINGS
583
TABLE I SUBCELLULAR LOCALIZATION OF DIHYDROFOLATE SYNTHETASE IN PEA SEEDLINGS
Fraction Homogenate Debris and nuclear Chloroplast Mitochondrial Microsomal Soluble
Protein (rag)
Enzyme activity (units)
2679 828 369 171 38 1190
253 31 39 102 0 57
Specific activity (units/mg protein)
Ratio (%)
0.094 0.037 0.106 0.597 0 0.048
100.0 12.3 15.4 40.3 0 22.5
Mitochondrial Extract. Six-day-old pea seedlings (100 kg) are homogenized with 100 liters of the isolation medium. The homogenate is squeezed through cotton cloth in a basket centrifuge. The green juice (130 liters) is kept for more than 2 hr, after which the precipitate is removed by decantation and the supernatant is centrifuged at 20,000 g for 30 min. The pellet is suspended in 10 liters of the suspending buffer. Fractionation with A m m o n i u m Sulfate. The suspension is centrifuged at 20,000 g for 30 rain. To the supernatant solution, solid ammonium sulfate is added to make 0.1 saturation with stirring at 5°. After being stirred for 30 min, the mixture is centrifuged at 20,000 g for 30 min. The precipitated protein recovered is dissolved in 2.4 liters of the suspending buffer. This solution is dialyzed against the same buffer, and the precipitate is discarded. The supernatant solution is divided into six 400-ml parts for convenient fractionation on DEAE-cellulose. Fractionation on DEAE-Cellulose. The dialyzed solution is applied on a DEAE-cellulose column (6 x 70 cm) which is previously equilibrated with 10 mM ammonium sulfate in the suspending buffer. After the protein solution is added, the column is washed with 2 liters of the same buffer. The column is developed by a linear gradient elution procedure in which the concentration of ammonium sulfate is increased from 10 to 200 mM in 2 liters of the buffer. Each collected fraction is analyzed for protein and for enzyme activity. Active fractions are combined and are recovered by precipitation with solid ammonium sulfate (0.6 saturation). The precipitated protein is dissolved in 100 ml of the suspending buffer, and the solution is divided into two 50-ml parts. Fractionation on Sephadex. Each 50-ml portion is applied to a Sephadex G-200 column (6 x 90 cm) which is previously equilibrated with 0.8
584
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[81]
M ammonium sulfate in 0. i M Tris-HCl buffer, pH 7.5, and 50 mM 2mercaptoethanol. Active fractions are combined and recovered by precipitation with solid ammonium sulfate (0.6 saturation). The precipitated protein is dissolved in the suspending buffer. The solution is again chromatographed on a Sephadex G-200 column (6 x 90 cm) under the same conditions. Active fractions are combined and are recovered by precipitation with solid ammonium sulfate (0.6 saturation). Fractionation on Hydroxylapatite. The dialyzed solution is applied to a hydroxylapatite column (4 x 15 cm) which is equilibrated with the suspending buffer. The column is developed with 50 mM potassium phosphate buffer, pH 7.5, containing 50 mM 2-mercaptoethanol after a total of 350 ml of 10 mM phosphate buffer has passed through the column. Fractions of 5 ml each are collected at a flow rate of 5 ml per 30 min. The active fractions in tubes 80-96 are pooled. The precipitate formed adding solid ammonium sulfate (0.6 saturation) is dissolved in 3 ml of the suspending buffer. Fractionation on Sephadex. The dialyzed solution is chromatographed on a Sephadex G-200 column (2.5 x 90 cm) under the conditions described above. A summary of the purification steps is given in Table II. Substrates Neither pteroic acid nor tetrahydropteroic acid can replace dihydropteroic acid as substrate. Other requirements for the reaction are L-gluTABLE II SUMMARY OF PURIFICATION OF DIHYDROFOLATE SYNTHETASE FROM PEA SEEDLINGS
Fraction
Total protein (mg)
Total activity (units)
Juice 4,128,000 477,300 Mitochondrial extract 698,000 321,556 10-35% Ammonium sulfate fraction 129,000 162,540 DEAE eluate 4,930 32,045 First Sephadex eluate 727 9,742 Second Sephadex eluate 100 8,560 Hydroxylapatite 45 8,190 Third Sephadex eluate 30 6,840
Specific activity (units/mg protein) 0.116 0.460 1.26 6.50 13.4 85.6 182 228
Overall Overall purification yield (-fold) (%) 1.0 4.0
100 67.3
10.9 56.1 115.5 738.0 1569.0 1965.6
34.1 6.7 2.0 1.8 1.7 1.4
[82]
FORMYLTETRAHYDROFOLATE SYNTHETASE
585
tamate, ATP, Mg2+, and a monovalent cation which is supplied as K +, NH4+, or Rb +. ATP cannot be replaced by other nucleotides tested. Mn 2+ can be utilized 143% as effectively as Mg2+ at each optimum concentration. The optimum concentrations of Mn 2+ and Mg2+ needed are 0.5 and 5.0 mM respectively. Li +, Na +, and Cs + have no effects on activity. ADP inhibits the reaction, but AMP has no effect on activity. Omission of 2mercaptoethanol from the complete reaction system results in decreasing the activity to 68%. Miscellaneous Properties Dihydrofolate synthetase operates optimally at pH 8.8 with significant activity in the range from pH 8.0 to 11.0. The apparent molecular weight of the enzyme is about 56,000. When the enzyme is stored at 0° for 3 months in l0 mM Tris-HCl buffer, pH 7.5, containing 2 M ammonium sulfate and 50 mM 2-mercaptoethanol, a dimer of the active enzyme, which has no activity, is formed. The sedimentation coefficient of the active enzyme at 20° is 3.9 S and that of the dimer is 7.5 S. The apparent Km wflues for dihydropteroic acid, L-glutamic acid, ATP, MgSO4, and MnSO4 were calculated to be 1.0 ~M, 1.5 raM, 0. l mM, 1. l mM, and 63 /.~M, respectively.
[82] P u r i f i c a t i o n a n d P r o p e r t i e s o f Formyltetrahydrofolate Synthetase By DANmL H. BUTTLAIRE (-)-Tetrahydrofolate + MgATP + formate ~(-)-Nl°-formyltetrahydrofolate + MgADP + P~
Occurrence Formyltetrahydrofolate synthetase (EC 6.3.4.3.) is widely distributed among prokaryotic and eukaryotic organisms.1 The specific activities of the enzyme in crude extracts of various organisms are summarized in Table I. The highest concentrations of the enzyme are found in bacterial sources, principally Micrococcus aerogenes, Clostridium cylindrosporum, Clostridium acidi-urici, and CIostridium thermoaceticum. The first three of these microorganisms possess the ability to ferment purines, I R. H . H i m e s a n d J. A . K . H a r m o n y , CRC Crit. Rev. Biochem. 1 , 5 0 1 (1973).
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
DIHYDROFOLATE
S Y N T H E T A S E F R O M PEA S E E D L I N G S
581
casei and S. f a e c i u m and suggests that its site of activity in the latter
organism is dihydrofolate synthetase. The eight compounds in the table and the substrate itself were all tested for inhibitory activity in their oxidized states. In no instance was inhibition observed. It is clearly mandatory that the 7- and 8-positions of the pteridine moiety be reduced to achieve binding of substrate and inhibitor to the enzyme. To confirm the kinetic behavior of the enzyme toward these newly synthesized pteroate analogs, independent experiments were carried out with a constant concentration of each drug and at least five levels of substrate concentration (i.e., dihydropteroic acid). From such velocity data, LineweaverBurk kinetic plots were obtained for each of the active compounds. The inhibitions exerted by compounds 1-4 of the table are found to be competitive in nature. The apparent K~ value for each compound is the result of at least five experiments, and it was observed experimentally that the kinetic data were reproducible to within 1%.
[81] I n t r a c e l l u l a r D i s t r i b u t i o n , P u r i f i c a t i o n , a n d Properties of Dihydrofolate Synthetase from Pea Seedlings By KAZUO IWAI, MASAMICHI IKEDA, and MASAHIRO KOBASHI
7,8-Dihydropteroate + L-glutamate+ATP Mn'+K+ (orM,'n )7,8-dihydrofolate+ADP +Pl
Assay Method Principle. The assay for dihydrofolate synthetase activity is based on the microbiological assay with Lactobacillus casei ATCC 7469 which is described by Iwai et al. 1 7,8-Dihydropteroic acid used as substrate is prepared from pteroic acid by reduction with sodium dithionite as described by Futterman ~ for reduction of folic acid to 7,8-dihydrofolic acid. Reagents
Tris-HCl buffer, 0.1 M, pH 8.8 MgSO4, 5 mM K2804, 50 mM L-Glutamic acid, 5 mM i K. lwai, O. Okinaka, and H. Yokomizo,Vitamins 35, 387 (1967). 2 S. Futterman,J. Biol. Chem. 228, 1031 (1957). METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
582
PTERIDINES, ANALOGS, AND PTER1N COENZYMES
[81]
ATP, 5 mM Dihydropteroic acid, 50 p~M 2-Mercaptoethanol, 50 mM Procedure. 3 The above reagents are added to a tube in a total volume of 1.0 ml. The reaction is started by the addition of the enzyme. After the incubation at 37° for 30 min, the reaction is stopped by heating the whole in a boiling water bath for 1 min. An aliquot of the reaction mixture is added to the single basal medium for folic acid in a final volume of 10 ml. Definition o f a Unit. One unit of enzyme is that amount required for the formation of 0.1 nmol of folate equivalent under the above conditions. Protein is determined by the method of Lowry et al.4 with bovine serum albumin as the standard. Isolation Procedure for Cell Particles 3 Fifty grams of 6-day-old pea seedlings (Pisum sativum L. var. Alaska) are homogenized in a mortar with 100 ml of isolation medium containing 0.5 M sucrose, 50 mM Tris-HCl buffer, pH 7.5, and 50 mM 2-mercaptoethanol. The homogenate is squeezed through four layers of gauze and then centrifuged by the modified method of Fujiwara and Iida 5 successively at 100 g for 5 min (debris and nuclei), at 1000 g for 12 rain (chloroplasts), at 20,000 g for 30 rain (broken chloroplasts and mitochondria), and at 105,000 g for 90 min (microsomes). The final supernatant is used as the soluble fraction. Each precipitated fraction is washed with the isolation medium and suspended in 10 mM Tris-HC1 buffer, pH 7.5, containing 50 mM 2-mercaptoethanol (the suspending buffer). The subcellular localization of the enzyme is given in Table I. Purification Procedure Principle. The enzyme can be partially stabilized in the presence of 0.8-2 M ammonium sulfate and 50 mM 2-mercaptoethanol, although the enzyme in cell-free extracts from whole pea seedling is quite unstable, e In contrast, the enzyme in the mitochondrial fraction is fairly stable and easily solubilized by more than 95% only when in the suspending buffer. 3 M. l k e d a and K. Iwai, J. Nutr. Sci. Vitaminol. 21, 1 (1975). 4 0 . H. L o w r y , N. J. R o s e b r o u g h , A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A. Fujiwara and S. Iida, J. Sci. Soil Manure, Jpn. 31, 467 (1960). e M. Ikeda and K. Iwai, Plant Cell Physiol. 11,639 (1970).
[81]
DIHYDROFOLATE SYNTHETASE FROM PEA SEEDLINGS
583
TABLE I SUBCELLULAR LOCALIZATION OF DIHYDROFOLATE SYNTHETASE IN PEA SEEDLINGS
Fraction Homogenate Debris and nuclear Chloroplast Mitochondrial Microsomal Soluble
Protein (rag)
Enzyme activity (units)
2679 828 369 171 38 1190
253 31 39 102 0 57
Specific activity (units/mg protein)
Ratio (%)
0.094 0.037 0.106 0.597 0 0.048
100.0 12.3 15.4 40.3 0 22.5
Mitochondrial Extract. Six-day-old pea seedlings (100 kg) are homogenized with 100 liters of the isolation medium. The homogenate is squeezed through cotton cloth in a basket centrifuge. The green juice (130 liters) is kept for more than 2 hr, after which the precipitate is removed by decantation and the supernatant is centrifuged at 20,000 g for 30 min. The pellet is suspended in 10 liters of the suspending buffer. Fractionation with A m m o n i u m Sulfate. The suspension is centrifuged at 20,000 g for 30 rain. To the supernatant solution, solid ammonium sulfate is added to make 0.1 saturation with stirring at 5°. After being stirred for 30 min, the mixture is centrifuged at 20,000 g for 30 min. The precipitated protein recovered is dissolved in 2.4 liters of the suspending buffer. This solution is dialyzed against the same buffer, and the precipitate is discarded. The supernatant solution is divided into six 400-ml parts for convenient fractionation on DEAE-cellulose. Fractionation on DEAE-Cellulose. The dialyzed solution is applied on a DEAE-cellulose column (6 x 70 cm) which is previously equilibrated with 10 mM ammonium sulfate in the suspending buffer. After the protein solution is added, the column is washed with 2 liters of the same buffer. The column is developed by a linear gradient elution procedure in which the concentration of ammonium sulfate is increased from 10 to 200 mM in 2 liters of the buffer. Each collected fraction is analyzed for protein and for enzyme activity. Active fractions are combined and are recovered by precipitation with solid ammonium sulfate (0.6 saturation). The precipitated protein is dissolved in 100 ml of the suspending buffer, and the solution is divided into two 50-ml parts. Fractionation on Sephadex. Each 50-ml portion is applied to a Sephadex G-200 column (6 x 90 cm) which is previously equilibrated with 0.8
584
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[81]
M ammonium sulfate in 0. i M Tris-HCl buffer, pH 7.5, and 50 mM 2mercaptoethanol. Active fractions are combined and recovered by precipitation with solid ammonium sulfate (0.6 saturation). The precipitated protein is dissolved in the suspending buffer. The solution is again chromatographed on a Sephadex G-200 column (6 x 90 cm) under the same conditions. Active fractions are combined and are recovered by precipitation with solid ammonium sulfate (0.6 saturation). Fractionation on Hydroxylapatite. The dialyzed solution is applied to a hydroxylapatite column (4 x 15 cm) which is equilibrated with the suspending buffer. The column is developed with 50 mM potassium phosphate buffer, pH 7.5, containing 50 mM 2-mercaptoethanol after a total of 350 ml of 10 mM phosphate buffer has passed through the column. Fractions of 5 ml each are collected at a flow rate of 5 ml per 30 min. The active fractions in tubes 80-96 are pooled. The precipitate formed adding solid ammonium sulfate (0.6 saturation) is dissolved in 3 ml of the suspending buffer. Fractionation on Sephadex. The dialyzed solution is chromatographed on a Sephadex G-200 column (2.5 x 90 cm) under the conditions described above. A summary of the purification steps is given in Table II. Substrates Neither pteroic acid nor tetrahydropteroic acid can replace dihydropteroic acid as substrate. Other requirements for the reaction are L-gluTABLE II SUMMARY OF PURIFICATION OF DIHYDROFOLATE SYNTHETASE FROM PEA SEEDLINGS
Fraction
Total protein (mg)
Total activity (units)
Juice 4,128,000 477,300 Mitochondrial extract 698,000 321,556 10-35% Ammonium sulfate fraction 129,000 162,540 DEAE eluate 4,930 32,045 First Sephadex eluate 727 9,742 Second Sephadex eluate 100 8,560 Hydroxylapatite 45 8,190 Third Sephadex eluate 30 6,840
Specific activity (units/mg protein) 0.116 0.460 1.26 6.50 13.4 85.6 182 228
Overall Overall purification yield (-fold) (%) 1.0 4.0
100 67.3
10.9 56.1 115.5 738.0 1569.0 1965.6
34.1 6.7 2.0 1.8 1.7 1.4
[82]
FORMYLTETRAHYDROFOLATE SYNTHETASE
585
tamate, ATP, Mg2+, and a monovalent cation which is supplied as K +, NH4+, or Rb +. ATP cannot be replaced by other nucleotides tested. Mn 2+ can be utilized 143% as effectively as Mg2+ at each optimum concentration. The optimum concentrations of Mn 2+ and Mg2+ needed are 0.5 and 5.0 mM respectively. Li +, Na +, and Cs + have no effects on activity. ADP inhibits the reaction, but AMP has no effect on activity. Omission of 2mercaptoethanol from the complete reaction system results in decreasing the activity to 68%. Miscellaneous Properties Dihydrofolate synthetase operates optimally at pH 8.8 with significant activity in the range from pH 8.0 to 11.0. The apparent molecular weight of the enzyme is about 56,000. When the enzyme is stored at 0° for 3 months in l0 mM Tris-HCl buffer, pH 7.5, containing 2 M ammonium sulfate and 50 mM 2-mercaptoethanol, a dimer of the active enzyme, which has no activity, is formed. The sedimentation coefficient of the active enzyme at 20° is 3.9 S and that of the dimer is 7.5 S. The apparent Km wflues for dihydropteroic acid, L-glutamic acid, ATP, MgSO4, and MnSO4 were calculated to be 1.0 ~M, 1.5 raM, 0. l mM, 1. l mM, and 63 /.~M, respectively.
[82] P u r i f i c a t i o n a n d P r o p e r t i e s o f Formyltetrahydrofolate Synthetase By DANmL H. BUTTLAIRE (-)-Tetrahydrofolate + MgATP + formate ~(-)-Nl°-formyltetrahydrofolate + MgADP + P~
Occurrence Formyltetrahydrofolate synthetase (EC 6.3.4.3.) is widely distributed among prokaryotic and eukaryotic organisms.1 The specific activities of the enzyme in crude extracts of various organisms are summarized in Table I. The highest concentrations of the enzyme are found in bacterial sources, principally Micrococcus aerogenes, Clostridium cylindrosporum, Clostridium acidi-urici, and CIostridium thermoaceticum. The first three of these microorganisms possess the ability to ferment purines, I R. H . H i m e s a n d J. A . K . H a r m o n y , CRC Crit. Rev. Biochem. 1 , 5 0 1 (1973).
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3